| On-line monitoring of apoptosis in insulin-secreting cells. | |
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MedLine Citation:
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PMID: 14633855 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Apoptosis was monitored in intact insulin-producing cells both with microfluorometry and with two-photon laser scanning microscopy (TPLSM), using a fluorescent protein based on fluorescence resonance energy transfer (FRET). TPLSM offers three-dimensional spatial information that can be obtained relatively deep in tissues. This provides a potential for future in vivo studies of apoptosis. The cells expressed a fluorescent protein (C-DEVD-Y) consisting of two fluorophores, enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), linked by the amino acid sequence DEVD selectively cleaved by caspase-3-like proteases. FRET between ECFP and EYFP in C-DEVD-Y could therefore be monitored on-line as a sensor of caspase-3 activation. The relevance of using caspase-3 activation to indicate beta-cell apoptosis was demonstrated by inhibiting caspase-3-like proteases with Z-DEVD-fmk and thereby showing that caspase-3 activation was needed for high-glucose-and cytokine-induced apoptosis in the beta-cell and for staurosporine-induced apoptosis in RINm5F cells. In intact RINm5F cells expressing C-DEVD-Y and in MIN6 cells expressing the variant C-DEVD-Y2, FRET was lost at 155 +/- 23 min (n = 9) and 257 +/- 59 min (n = 4; mean +/- SE) after activation of apoptosis with staurosporine (6 micromol/l), showing that this method worked in insulin-producing cells. |
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Authors:
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Martin Köhler; Sergei V Zaitsev; Irina I Zaitseva; Barbara Leibiger; Ingo B Leibiger; Mikael Turunen; Iouri L Kapelioukh; Linda Bakkman; Ioulia B Appelskog; Jacques Boutet de Monvel; Gabriela Imreh; Per-Olof Berggren |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Diabetes Volume: 52 ISSN: 0012-1797 ISO Abbreviation: Diabetes Publication Date: 2003 Dec |
Date Detail:
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Created Date: 2003-11-24 Completed Date: 2004-01-09 Revised Date: 2007-11-14 |
Medline Journal Info:
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Nlm Unique ID: 0372763 Medline TA: Diabetes Country: United States |
Other Details:
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Languages: eng Pagination: 2943-50 Citation Subset: AIM; IM |
Affiliation:
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Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, Stockholm, Sweden. martin.kohler@molmed.ki.se |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Animals Apoptosis* Bacterial Proteins Caspase 3 Caspases / metabolism Cells, Cultured Enzyme Precursors / metabolism Fluorescence Resonance Energy Transfer Fluorometry Green Fluorescent Proteins Indicators and Reagents Insulin / secretion* Internet* Islets of Langerhans / secretion* Luminescent Proteins Mice Microscopy, Confocal Monitoring, Physiologic* Obesity / genetics, metabolism, physiopathology* Photons |
| Grant Support | |
ID/Acronym/Agency:
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DK-58508/DK/NIDDK NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Bacterial Proteins; 0/Enzyme Precursors; 0/Indicators and Reagents; 0/Luminescent Proteins; 0/yellow fluorescent protein, Bacteria; 11061-68-0/Insulin; 147336-22-9/Green Fluorescent Proteins; EC 3.4.22.-/Casp3 protein, mouse; EC 3.4.22.-/Caspase 3; EC 3.4.22.-/Caspases |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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